Aim: Genus Anaplasma is of veterinary and public health importance, and its members utilize ruminants as key hosts in their epidemiology. To date, information about the occurrence and molecular identity of Anaplasma phagocytophilum and other Anaplasma species in Saudi Arabian goats is scarce. This study aimed to molecularly detect and characterize zoonotic A. phagocytophilum and other Anaplasma spp. in goats from Taif District, KSA.

Materials and Methods: Blood samples collected from 67 goats were polymerase chain reaction tested using common and A. phagocytophilum-specific primers targeting 16S rRNA and msp4 genes, respectively. Amplicons of common reactions were purified, sequenced, and analyzed.

Results: Six goats yielded positive results with common primers, whereas all animals proved negative for A. phagocytophilum. Analysis of the two successfully sequenced amplicons revealed the presence of a variant strain of Anaplasma ovis (99.52% ID) and a new Anaplasma organism, which was clustered with Anaplasma bovis (95.9% ID) and Aegyptianella pullorum (94.99% ID) and distinctly separated from all other recognized species of the genus Anaplasma.

Conclusion: The tested goats proved negative for A. phagocytophilum; however, we could not confirm that the area is pathogen free. A variant strain of A. ovis and a putative novel Anaplasma spp. were reported raising the concern of veterinary and zoonotic potential. Other genes should be sequenced and analyzed for complete identification of the detected organisms.

Keywords: Anaplasma ovis, Anaplasma phagocytophilum, goats, phylogeny, Saudi Arabia.

1. Dumler JS, Barbet AF, Bekker CP, Dasch GA, Palmer GH, Ray SC, et al. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: Unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and "HGE agent" as subjective synonyms of Ehrlichia phagocytophila. Int J Syst Evol Microbiol 2001;51:2145-65. [Crossref] [PubMed]

2. Rikihisa Y, Zhang C, Christensen BM. Molecular characterization of Aegyptianella pullorum (Rickettsiales, Anaplasmataceae). J Clin Microbiol 2003;41:5294-7. [Crossref] [PubMed] [PMC]

3. Li H, Zheng YC, Ma L, Jia N, Jiang BG, Jiang RR, et al. Human infection with a novel tick-borne Anaplasma species in China: A surveillance study. Lancet Infect Dis 2015;15:663-70. [Crossref]

4. Yang J, Li Y, Liu Z, Liu J, Niu Q, Ren Q, et al. Molecular detection and characterization of Anaplasma spp. In sheep and cattle from Xinjiang, northwest China. Parasit Vectors 2015;8:108. [Crossref] [PubMed] [PMC]

5. Inokuma H, Terada Y, Kamio T, Raoult D, Brouqui P. Analysis of the 16S rRNA gene sequence of Anaplasma centrale and its phylogenetic relatedness to other Ehrlichiae. Clin Diagn Lab Immunol 2001;8:241-4. [Crossref] [PubMed] [PMC]

6. Byaruhanga C, Collins NE, Knobel DL, Khumalo ZTH, Chaisi ME, Oosthuizen MC, et al. Molecular detection and phylogenetic analysis of Anaplasma marginale and Anaplasma centrale amongst transhumant cattle in North-Eastern Uganda. Ticks Tick Borne Dis 2018;9:580-8. [Crossref] [PubMed]

7. Renneker S, Abdo J, Salih DE, Karagenc T, Bilgic H, Torina A, et al. Can Anaplasma ovis in small ruminants be neglected any longer? Transbound Emerg Dis 2013;60 Suppl 2:105-12. [Crossref] [PubMed]

8. Kocan KM, de la Fuente J, Blouin EF, Coetzee JF, Ewing SA. The natural history of Anaplasma marginale. Vet Parasitol 2010;167:95-107. [Crossref] [PubMed]

9. Adjou Moumouni PF, Aboge GO, Terkawi MA, Masatani T, Cao S, Kamyingkird K, et al. Molecular detection and characterization of Babesia bovis, Babesia bigemina, Theileria species and Anaplasma marginale isolated from cattle in Kenya. Parasit Vectors 2015;8:496. [Crossref] [PubMed] [PMC]

10. Battilani M, De Arcangeli S, Balboni A, Dondi F. Genetic diversity and molecular epidemiology of Anaplasma. Infect Genet Evol 2017;49:195-211. [Crossref] [PubMed]

11. Rjeibi MR, Ayadi O, Rekik M, Gharbi M. Molecular survey and genetic characterization of Anaplasma centrale, A. marginale and A. bovis in cattle from Algeria. Transbound Emerg Dis 2018;65:456-64. [Crossref] [PubMed]

12. Kuttler KL. Anaplasma infections in wild and domestic ruminants: A review. J Wildl Dis 1984;20:12-20. [Crossref] [PubMed]

13. Friedhoff KT. Tick-borne diseases of sheep and goats caused by Babesia, Theileria or Anaplasma spp. Parasitologia 1997;39:99-109.

14. Tibbitts T, Goff W, Foreyt W, Stiller D. Susceptibility of two rocky mountain bighorn sheep to experimental infection with Anaplasma ovis. J Wildl Dis 1992;28:125-9. [Crossref] [PubMed]

15. Lee SH, Mossaad E, Ibrahim AM, Ismail AA, Moumouni PF, Liu M, et al. Detection and molecular characterization of tick-borne pathogens infecting sheep and goats in Blue Nile and West Kordofan states in Sudan. Ticks Tick Borne Dis 2018;9:598-604. [Crossref] [PubMed]

16. Chochlakis D, Ioannou I, Tselentis Y, Psaroulaki A. Human anaplasmosis and Anaplasma ovis variant. Emerg Infect Dis 2010;16:1031-2. [Crossref] [PubMed] [PMC]

17. Hosseini-Vasoukolaei N, Oshaghi MA, Shayan P, Vatandoost H, Babamahmoudi F, Yaghoobi-Ershadi MR, et al. Anaplasma infection in ticks, livestock and human in Ghaemshahr, Mazandaran Province, Iran. J Arthropod Borne Dis 2014;8:204-11.

18. Said MB, Belkahia H, Messadi L. Anaplasma spp. In North Africa: A review on molecular epidemiology, associated risk factors and genetic characteristics. Ticks Tick Borne Dis 2018;9:543-55. [Crossref] [PubMed]

19. Vieira FT, Acosta IC, Martins TF, Filho JM, Krawczak FD, Barbieri AR, et al. Tick-borne infections in dogs and horses in the state of Espirito Santo, Southeast Brazil. Vet Parasitol 2018;249:43-8. [Crossref] [PubMed]

20. Bastos AD, Mohammed OB, Bennett NC, Petevinos C, Alagaili AN. Molecular detection of novel Anaplasmataceae closely related to Anaplasma platys and Ehrlichia canis in the dromedary camel (Camelus dromedarius). Vet Microbiol 2015;179:310-4. [Crossref] [PubMed]

21. Belkahia H, Said MB, Sayahi L, Alberti A, Messadi L. Detection of novel strains genetically related to Anaplasma platys in Tunisian one-humped camels (Camelus dromedarius). J Infect Dev Ctries 2015;9:1117-25. [Crossref] [PubMed]

22. Li Y, Yang J, Chen Z, Qin G, Li Y, Li Q, et al. Anaplasma infection of Bactrian camels (Camelus bactrianus) and ticks in Xinjiang, China. Parasit Vectors 2015;8:313. [Crossref] [PubMed] [PMC]

23. Zobba R, Anfossi AG, Parpaglia ML, Dore GM, Chessa B, Spezzigu A, et al. Molecular investigation and phylogeny of Anaplasma spp. In Mediterranean ruminants reveal the presence of neutrophil-tropic strains closely related to A. platys. Appl Environ Microbiol 2014;80:271-80. [Crossref] [PubMed] [PMC]

24. Liu Z, Ma M, Wang Z, Wang J, Peng Y, Li Y, et al. Molecular survey and genetic identification of Anaplasma species in goats from central and Southern China. Appl Environ Microbiol 2012;78:464-70. [Crossref] [PubMed] [PMC]

25. Arraga-Alvarado CM, Qurollo BA, Parra OC, Berrueta MA, Hegarty BC, Breitschwerdt EB, et al. Case report: Molecular evidence of Anaplasma platys infection in two women from Venezuela. Am J Trop Med Hyg 2014;91:1161-5. [Crossref] [PubMed] [PMC]

26. Noaman V, Shayan P. Molecular detection of Anaplasma bovis in cattle from central part of Iran. Vet Res Forum 2010;1:117-22.

27. Ooshiro M, Zakimi S, Matsukawa Y, Katagiri Y, Inokuma H. Detection of Anaplasma bovis and Anaplasma phagocytophilum from cattle on Yonaguni Island, Okinawa, Japan. Vet Parasitol 2008;154:360-4. [Crossref] [PubMed]

28. Jilintai, Seino N, Hayakawa D, Suzuki M, Hata H, Kondo S, et al. Molecular survey for Anaplasma bovis and Anaplasma phagocytophilum infection in cattle in pastureland where sika deer appear in Hokkaido, Japan. Jpn J Infect Dis 2009;62:73-5.

29. Chen SM, Dumler JS, Bakken JS, Walker DH. Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease. J Clin Microbiol 1994;32:589-95.

30. Jahfari S, Coipan EC, Fonville M, van Leeuwen AD, Hengeveld P, Heylen D, et al. Circulation of four Anaplasma phagocytophilum ecotypes in Europe. Parasit Vectors 2014;7:365. [Crossref] [PubMed] [PMC]

31. de la Fuente J, Estrada-Pena A, Cabezas-Cruz A, Kocan KM. Anaplasma phagocytophilum uses common strategies for infection of ticks and vertebrate hosts. Trends Microbiol 2016;24:173-80. [Crossref] [PubMed]

32. Ochirkhuu N, Konnai S, Odbileg R, Murata S, Ohashi K. Molecular epidemiological survey and genetic characterization of Anaplasma species in Mongolian livestock. Vector Borne Zoonotic Dis 2017;17:539-49. [Crossref] [PubMed]

33. Zhan L, Cao WC, Jiang JF, Zhang XA, Wu XM, Zhang WY, et al. Anaplasma phagocytophilum in livestock and small rodents. Vet Microbiol 2010;144:405-8. [Crossref] [PubMed]

34. Dumler JS, Choi KS, Garcia-Garcia JC, Barat NS, Scorpio DG, Garyu JW, et al. Human granulocytic anaplasmosis and Anaplasma phagocytophilum. Emerg Infect Dis 2005;11:1828-34. [Crossref] [PubMed] [PMC]

35. Dahmani M, Davoust B, Benterki MS, Fenollar F, Raoult D, Mediannikov O, et al. Development of a new PCR-based assay to detect Anaplasmataceae and the first report of Anaplasma phagocytophilum and Anaplasma platys in cattle from Algeria. Comp Immunol Microbiol Infect Dis 2015;39:39-45. [Crossref] [PubMed]

36. M'ghirbi Y, Beji M, Oporto B, Khrouf F, Hurtado A, Bouattour A, et al. Anaplasma marginale and A. phagocytophilum in cattle in Tunisia. Parasit Vectors 2016;9:556. [Crossref] [PubMed] [PMC]

37. Yang J, Liu Z, Niu Q, Liu J, Han R, Liu G, et al. Molecular survey and characterization of a novel Anaplasma species closely related to Anaplasma capra in ticks, Northwestern China. Parasit Vectors 2016;9:603. [Crossref] [PubMed] [PMC]

38. Cicala F, Moore JD, Caceres-Martinez J, Rio-Portilla MA, Hernandez-Rodriguez M, Vasquez-Yeomans R, et al. Multigenetic characterization of "Candidatus Xenohaliotis californiensis". Int J Syst Evol Microbiol 2017;67:42-9. [Crossref] [PubMed]

39. Dahmani M, Davoust B, Tahir D, Raoult D, Fenollar F, Mediannikov O, et al. Molecular investigation and phylogeny of Anaplasmataceae species infecting domestic animals and ticks in Corsica, France. Parasit Vectors 2017;10:302. [Crossref] [PubMed] [PMC]

40. Ehounoud CB, Yao KP, Dahmani M, Achi YL, Amanzougaghene N, Kacou N'Douba A, et al. Multiple pathogens including potential new species in tick vectors in Cote d'Ivoire. PLoS Negl Trop Dis 2016;10:e0004367. [Crossref] [PubMed] [PMC]

41. Eshoo MW, Carolan HE, Massire C, Chou DM, Crowder CD, Rounds MA, et al. Survey of Ixodes pacificus ticks in California reveals a diversity of microorganisms and a novel and widespread Anaplasmataceae species. PLoS One 2015;10:e0135828. [Crossref] [PubMed] [PMC]

42. Guo WP, Tian JH, Lin XD, Ni XB, Chen XP, Liao Y, et al. Extensive genetic diversity of Rickettsiales bacteria in multiple mosquito species. Sci Rep 2016;6:38770. [Crossref] [PubMed] [PMC]

43. Li Y, Chen Z, Liu Z, Liu J, Yang J, Li Q, et al. Molecular survey of Anaplasma and Ehrlichia of red deer and sika deer in Gansu, China in 2013. Transbound Emerg Dis 2016;63:e228-36. [Crossref] [PubMed]

44. Qin XR, Han FJ, Luo LM, Zhao FM, Han HJ, Zhang ZT, et al. Anaplasma species detected in Haemaphysalis longicornis tick from China. Ticks Tick Borne Dis 2018;9:840-3. [Crossref] [PubMed]

45. Ghafar MW, Shobrak MY Molecular detection and characterization of Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis, in some animals suspected to be competent reservoirs in Taif district, Kingdom of Saudi Arabia. Life Sci J 2014;11:63-9.

46. Taha HA, Shoman SA, Alhadlag NM. Molecular and serological survey of some hemoprotozoan, rickettsial and viral diseases of small ruminants from al-Madinah al Munawarah, KSA. Trop Biomed 2015;32:511-23.

47. Kocan AA, Levesque GC, Whitworth LC, Murphy GL, Ewing SA, Barker RW, et al. Naturally occurring Ehrlichia chaffeensis infection in coyotes from Oklahoma. Emerg Infect Dis 2000;6:477-80. [Crossref] [PubMed] [PMC]

48. Murphy GL, Ewing SA, Whitworth LC, Fox JC, Kocan AA. A molecular and serologic survey of Ehrlichia canis, E. chaffeensis, and E. ewingii in dogs and ticks from Oklahoma. Vet Parasitol 1998;79:325-39. [Crossref]

49. de la Fuente J, Massung RF, Wong SJ, Chu FK, Lutz H, Meli M, et al. Sequence analysis of the msp4 gene of Anaplasma phagocytophilum strains. J Clin Microbiol 2005;43:1309-17. [Crossref] [PubMed] [PMC]

50. Katoh K, Rozewicki J, Yamada KD. MAFFT online service: Multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform 2017: 1-7. [Crossref] [PubMed]

51. Saitou N, Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol 1987;4:406-25.

52. Ismael AB, Swelum AA, Khalaf AF, Alowaimer AN. First evidence of natural anaplasmosis in Camelus dromedarius in Saudi Arabia. J Camel Pract Res 2016;23:95-100. [Crossref]

53. El-Metenawy TM. Prevalence of blood parasites among cattle at the central area of Saudi Arabia. Vet Parasitol 2000;87:231-6. [Crossref]

54. Al-Khalifa MS, Hussein HS, Diab FM, Khalil GM. Blood parasites of livestock in certain regions in Saudi Arabia. Saudi J Biol Sci 2009;16:63-7. [Crossref] [PubMed] [PMC]

55. Al-Gharban HA, Al-Taee HS. Seroclinical diagnosis of Anaplasma marginale bacteria in carrier Arabian one-humped camels. Bas J Vet Res 2016;15:346-59.

56. Lew AE, Gale KR, Minchin CM, Shkap V, de Waal DT. Phylogenetic analysis of the erythrocytic Anaplasma species based on 16S rDNA and groEL (HSP60) sequences of A. marginale, A. centrale, and A. ovis and the specific detection of A. centrale vaccine strain. Vet Microbiol 2003;92:145-60. [Crossref]

57. Adekambi T, Shinnick TM, Raoult D, Drancourt M. Complete rpoB gene sequencing as a suitable supplement to DNA-DNA hybridization for bacterial species and genus delineation. Int J Syst Evol Microbiol 2008;58:1807-14. [Crossref] [PubMed]

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